Isolation of camelid single-domain antibodies against native protins using recombinant multivalent peptide ligands

From National Research Council Canada

DOIResolve DOI: https://doi.org/10.1007/978-1-4939-2999-3_16
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Name affiliation
  1. National Research Council of Canada. Human Health Therapeutics
FormatText, Book chapter
Book titlePeptide Antibodies: Methods and Protocols
Series titleMethods in Molecular Biology; no. 1348
ISSN1064-3745
ISBN978-1-4939-2998-6
978-1-4939-2999-3
Pages167–189
Subjectsingle-domain antibody; VHH; camelid; peptide; phage display; NGS
Abstract
Generation of antibodies against desired epitopes on folded proteins may be hampered by various characteristics of the target protein, including antigenic and immunogenic dominance of irrelevant epitopes and/or steric occlusion of the desired epitope. In such cases, peptides encompassing linear epitopes of the native protein represent attractive alternative reagents for immunization and screening. Peptide antigens are typically prepared by fusing or conjugating the peptide of interest to a carrier protein. The utility of such antigens depends on many factors including the peptide's amino acid sequence, display valency, display format (synthetic conjugate vs. recombinant fusion) and characteristics of the carrier. Here we provide detailed protocols for: (i) preparation of DNA constructs encoding peptides fused to verotoxin (VT) multimerization domain; (ii) expression, purification and characterization of the multivalent peptide-VT ligands; (iii) concurrent panning of a non-immune phage-displayed camelid VHH library against the peptide-VT ligands and native protein; and (iv) identification of VHHs enriched via panning using next-generation sequencing techniques. These methods are simple, rapid and can be easily adapted to yield cusstom peptide-VT ligands that appear to maintain the antigenetic structures of the peptide. However, we caution that peptide sequences should be chosen with great care, taking into account structural, immunological, and biophysical information on the protein of interest.
Publication date
LanguageEnglish
Peer reviewedYes
IdentifierNRC-HHT-53286
NPARC number21277512
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